The following is offered as background information only and is not intended nor admitted to be prior art to the present invention.
The ability to detect DNA sequence variances in an organism's genome has become an important tool in the diagnosis of diseases and disorders and in the prediction of response to potential therapeutic regimes. It is becoming increasingly possible, using early variance detection, to diagnose and treat, even prevent, a disorder before it has physically manifested itself. Furthermore, variance detection can be a valuable research tool in that it may lead to the discovery of genetic bases for disorders the cause of which were hitherto unknown or thought to be other than genetic.
It is estimated that sequence variations in human DNA occur with a frequency of about 1 in 100 nucleotides when 50 to 100 individuals are compared. Nickerson, D. A., Nature Genetics, 1998, 223–240. This translates to as many as 30 million variances in the human genome. However, very few of these variances have any effect on the physical well-being of humans. Detecting these 30 million variances and then determining which of them are relevant to human health is clearly a formidable task.
Once the DNA sequence of a DNA segment; e.g., a gene, a cDNA or, on a larger scale, a chromosome or an entire genome, has been determined, the existence of sequence variances in that DNA segment among members of the same species can be explored. Complete DNA sequencing is the definitive procedure for accomplishing this task. However, current DNA sequencing technology is costly, time consuming and, in order to assure accuracy, highly redundant. Most sequencing projects require a 5- to 10-fold coverage of each nucleotide to reach an acceptable error rate of 1 in 2,000 to 1 in 10,000 bases. In addition, DNA sequencing is an inefficient way to detect variances. A variance between two copies of a gene, for example when two chromosomes are being compared, may occur as infrequently as one in 1,000 or more bases. Thus, only a small segment of the gene is of interest. If full sequencing is employed, a tremendous number of nucleotides have to be sequenced to arrive at the desired information contained in that segment. For example, to compare ten versions of a 3,000 nucleotide DNA sequence for the purpose of detecting four variances among them, even if only 2-fold redundancy is employed (each strand of the double-stranded 3,000 nucleotide DNA segment from each individual is sequenced once), 60,000 nucleotides would have to be sequenced (10×3,000×2). In addition, sequencing problems are often encountered that can require additional runs with new primers. Thus, as many as 100,000 nucleotides might have to be sequenced to determine four variances.
What is needed is a rapid, inexpensive, yet accurate method to identify variances such as SNPs among related polynucleotides. The present invention provides such a method and materials for its implementation.